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1. Digestion of Plasmid
The adenoviral vector is part of a bacterial plasmid. In order to release the adenoviral ends, it is necessary to digest with Pac I. The best method is to digest 12 µg of the plasmid in 100 µl total volume for 3 hours. Then the enzyme is inactivated through heating to 65 °C for 20 minutes. The rest can be stored at -20 °C.
2. Transformation of 293A Cells
Here 293A cells are needed. Alternatively, you can use 293 cells or 911 cells. It is important that they produce the adeno-E1 proteins since they are deleted in the vector.
(1) Split the 293A cells that they are 50% confluent. The 293A cells usually are split 1:3 into small 25 cm2 flasks.
(2) Transform the 293A cells using Lipofectamine 2000. Alternatively, CaPO4 can be used, but Lipofectamine gives more constant results.
(3) Refresh the medium on the 293A cells. You can already check transformation efficiency. If you see some green cells you are in the game, the more the better, 30% would be quite good.
(4) Split the 293 cells. The complete content of the small 25 cm2 flask is spread on a bigger 175 cm2 flask. Then, the cells are widely spread.
(5) Refresh the medium on the 293A cells. When there are holes in the cell layer or the cell layer detaches from the plastic, then harvest the cells.
(6) To harvest cell, you need to flush off the cells and freeze them with the medium at -80°C. The harvested medium/cell mix can be freeze/thawed and aliquoted. This is the F1 adenovirus stock. You can use it directly to infect new 293A cells.
3. Amplification of Adenoviral Vectors
(1) Prepare 175 cm2 flasks with 293A cells.
(2) The prepared big 175 cm2 flasks should be 80% confluent. Discard the old medium.
Note: If you use a F1 stock, it may not have a sufficient concentration of infectious particles, thus, amplification is necessary. Add 15 ml F1 adenovirus stock and incubate for 30 min. Then add 10 ml of fresh medium to the F1 adenovirus stock.
(3) Incubate at least for 3 hours, but not overnight. Remove the adenoviral vector mixture and replace it by fresh medium.
(4) The cells may detach 48-72 hours post infection, which depends on the concentration of adenoviral vectors. Harvest the cells, and the harvested medium/cell mix is freeze/thawed and aliquoted as before. This is the F2 adenovirus stock.
(5) Repeat this procedure till a good concentration is reached and the cells detach not after day 4 post-infection.